First report on the presence of huanglongbing vectors (Diaphorina citri and Trioza erytreae) in Ghana

As significant threats to global citrus production, Diaphorina citri (Kuwayama; Hemiptera: Psyllidae) and Trioza erytreae (Del Guercio; Hemiptera: Triozidae) have caused considerable losses to citrus trees globally. Diaphorina citri vectors “Candidatus Liberibacter asiaticus” and “Ca. L. americanus”, whereas T. erytreae transmits “Ca. L. africanus” and “Ca. L. asiaticus”, the pathogens responsible for citrus greening disease or Huanglongbing (HLB). Though HLB is a destructive disease of citrus wherever it occurs, information on the occurrence and geographical distribution of its vectors in Africa is limited. In recent surveys to determine if HLB vectors are present in Ghana, we observed eggs, nymphs, and adults of insects suspected to be D. citri and T. erytreae. Using morphological traits and DNA analyses, the identity of the suspected insects was confirmed to be D. citri and T. erytreae. Individuals of D. citri and T. erytreae were examined using qPCR for CLaf, CLam, and CLas, but none of them tested positive for any of the Liberibacter species. Herein we report, for the first time, the presence of D. citri and T. erytreae in Ghana (West Africa). We discuss the implications of this new threat to the citrus industry to formulate appropriate management strategies.

www.nature.com/scientificreports/ The development of the vectors goes through five nymphal instars. The adults of D. citri have speckled brown wings and measure between 2.7 and 3.3 mm in length 19 . The females of D. citri may lay a mean of 40 eggs per flush shoot per day 20 . The freshly laid eggs of D. citri are pale but change into yellow and orange with two unique red eye spots at maturity 19 . Diaphorina citri lays on tips of developing shoots on and between unfurling leaves 21 . Trioza erytreae is about 2.40 mm long, with females being slightly larger than males 18 . Trioza erytreae adults start pale but gradually darken afterwards to a light brown. Trioza erytreae female lays eggs on the margins and along the midribs of young and tender leaves 22 . The average number of eggs laid by T. erytreae females per day ranges from 17 in the cold to 39 in the warm seasons, respectively 23 . When feeding on leaves, adults rest at a body angle of about 35° and 45° for T. erytreae and D. citri, respectively 12,19 .
Both D. citri and T. erytreae cause direct and indirect damage to citrus. Feeding activities of D. citri nymphs and adults lead to substantial uptake of plant sap and may induce sooty mold development due to honeydew production on infested flush shoots. Sooty mold affects the photosynthetic activities of the citrus trees, thereby reducing their productivity 24 . Direct foliage feeding by T. erytreae results in stunted and seriously deformed leaves showing signs of pit-like galls 23 . Although direct feeding damage of psyllids may result in a loss of plant vigor, indirect damage via vectoring of HLB pathogens is the most economically important. All over the world, HLB is caused by the phloem-limited bacteria "Ca. L. asiaticus" (CLas) and Ca. Liberibacter americanus (CLam) transmitted by D. citri 25,26 . In contrast, in Africa, especially East and South Africa, T. erytreae represents an economically significant threat to the citrus industry because of its ability to vector "Ca. Liberibacter africanus" (CLaf), the causal agent of the African HLB. However, a recent study by Ajene et al. 27 showed field populations of T. erytreae carrying CLas, and Reunaud et al. 28 demonstrated that it also can efficiently transmit CLas. Unlike the HLB caused by CLaf, the HLB caused by CLas is the most destructive pathogen of all citrus in the world 28 .
D. citri have the ability to fly both long and short distances. However, its propensity to engage in longdistance flights, as well as the length and distance flown, are all affected by high heat conditions, regardless of the humidity level 29 . Nevertheless, the most extended flight distance and duration are shorter in females than males 30 . Additionally, D. citri routinely migrates between managed and unmanaged groves, covering a distance of about 60-100 m toward managed groves 31 . Because D. citri eggs are laid solely on young flush and nymphs develop primarily on young plant parts, population variations of this pest are highly correlated with the growth of new and young flush of the host plant 32,33 . According to Catling 23 , T. erytreae has weak dispersal powers and are incapable of sustaining flight for long hours. In contrast, Samways and Manicom 34 , showed that T. erytreae had a good dispersal ability and could invade orchards to locate new flush shoots.
Citrus is one of the most widely grown fruits worldwide due to its high demand and positive impact on food and nutritional security 35 . In terms of global trade value, it is one of the most valuable fruits 36 . The commonly known species of commercial importance are sweet oranges (Citrus sinensis), lemons (C. limon), limes (C. aurantifolia), grapefruits (C. paradisi), and tangerines (C. reticulata). Sweet oranges are the major species grown globally and represent more than half of the global citrus output. Annual global production of citrus was estimated at 158 million tonnes in 2020, with sweet oranges contributing to more than half of the world's total 37 . Africa produced 9,756,176 metric tons of sweet oranges from an estimated 514,573 ha in 2020, while production in Ghana was at 697,637 metric tons from an estimated 17,983 ha 37 . The health benefits of sweet oranges and other citrus fruits are well documented, especially in providing vitamin C, carotenoids, and polyphenols. Most citrus fruits in many countries, including Ghana, are consumed locally as fresh products 38 .
Early detection of D. citri and T. erytreae would help develop strategies and manage the pests and diseases they transmit, slowing down their spread and preventing them from becoming established. Despite reports of the presence of the invasive and deadly D. citri in neighboring Nigeria and Benin 10,11 , there have been no previous investigations to determine the existence of D. citri in Ghana. Given the economic importance of citrus production in Ghana, we conducted surveys for citrus commodity pests in Ghana to determine the presence of D. citri and T. erytreae and, if found, to test the samples for Liberibacter species.

Materials and methods
Sample collection. To establish the presence of D. citri and T. erytreae in Ghana, surveys of residential areas and citrus orchards in the Volta Region, Ghana (Table 1), were conducted from April to November 2022. The main roads in each town were chosen for sampling. All encountered Citrus and Murraya species were visually inspected for the presence of D. citri and T. erytreae.
Each backyard hedge (Fig. S1) with a host plant was visually examined for D. citri adults and symptomatic leaves (Fig. S2). Expanding flush shoots and at high densities on the stems are where D. citri nymphs are attached (Fig. S3). In contrast, T. erytreae nymphs are usually found on the underside of the leaves (Fig. S4). To collect the nymphal stages from the plants, we thoroughly searched for the immature stages on the stems and new tender shoots. Inspection of host plant was further aided by feeding damage, such as ' epinasty' , and distortion of young and fragile leaves due to the sap-feeding of D. citri nymphs and adults, and the presence of the white waxy excretions from the nymphs (D. citri) 10,19 . In addition, T. erytreae pit-like galls were used to detect the presence of the triozid 18 . In case adult insects were found, they were aspirated into plastic vials containing 95% ethanol, and nymphs were brushed into similar vials using a fine camel brush. The host plant observed was recorded for each location. Moreover, the coordinates of each sample location were recorded using a handheld Garmin eTrex ® 32x device. www.nature.com/scientificreports/ TAMUK-Entomology CC's laboratory, where they were compared to preserved reference voucher specimens. We used the morphological characteristics reported by Mead 4 , Yang 39 , and OEPP/EPPO 40 to positively identify D. citri nymphs and adults. Moreover, Aidoo et al. 18 and Cocuzza et al. 12 , reports on T. erytreae morphometry were used for their identification. The voucher specimens were kept at TAMUK-CC and the Central Laboratory of the University of Environment and Sustainable Development, Somanya, Ghana.

Identification
Nucleic acid isolation and PCR. In this study, we used the method described by Dellaporta et al. 41 to extract total nucleic acids (TNA) from both adults and nymphs of the psyllids. NanoDrop 2000 series spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) was used to measure the concentration and purity of the TNA extracts before they were frozen at 20 °C for later use. Two microliters (uL) of each sample was utilised as template in a 25 μL polymerase chain reaction (PCR) using the PrimeSTAR GXLDNA Polymerase, its recommended reagents, and the Rapid Protocol (Takara Bio USA, Inc., Mountain View, CA). We targeted 821-bp and 708-bp segments of the mtCOI coding region in the D. citri 42 and T. erytreae 43 using the primer pair DCITRI COI-L and DCITRI COI-R and Te-6U30 and Te-720L26, respectively. Positive controls consisted of DNA samples taken from TAMUK-lab-reared CC's ACPs. The 100-2000 bp Wide-Range DNA Ladder (Takara Bio USA, Inc.) and the amplified products were run on ethidium bromide-stained 1% agarose gels and then visualised using a UV-transilluminator.
Cloning and sequencing. Zymoclean Gel DNA Recovery Kit was used to cut out and gel-elute the appropriate size DNA bands from the sample and the target (Zymo Research, Irvine, CA). After purification, the recovered DNA was cloned into the pJET1.2/blunt vector one at a time using the CloningJET PCR Kit (Thermo Fisher Scientific). Transforming chemically competent DH5 Escherichia coli cells with the ligation products yielded two to three plasmids per cloned DNA amplicon that were PCR-verified to be the correct size (Sigma-Aldrich, St. Louis, MO). By using the Sanger sequencing technique and primers designated as pJET1.2 F and pJET1.2 R, each plasmid sample was sequenced in both directions (ELIM BIOPHARM, Hayward, CA, USA).
Bioinformatic analysis. The pJET1.2 vector sequences were removed using VecScreen (https:// www. ncbi. nlm. nih. gov/ tools/ vecsc reen/). Each sample's forward and reverse sequences were entered into the CAP contig assembly function of the BioEdit software 44 to generate a consensus sequence. To determine which species each consensus sequence belonged to, BLASTn analysis 45 was performed on all the sequences of the psyllids. Multiple sequence alignments were generated between the sequences derived in this study and those obtained from GenBank using the MUltiple Sequence Comparison by Log-Expectation alignment program (http:// www. ebi. ac. uk/ Tools/ msa/ muscle/). The sequences were chosen because they are representative of the diversity of the taxonomic groups studied. The sequence identity matrices and phylogenetic analyses were calculated using the maximum likelihood approach in MEGA version 7.0 46 , which was applied to the gene-specific alignment data. Instead of using tables of pairwise sequence identity scores, which are commonly used for this purpose, it is recommended to use the Sequence Demarcation Tool (SDT), which displays pairwise identity scores using a color-coded matrix 47 . This makes it easier to gain insights into the overall relationships between sequences in a dataset. Moreover, we calculated the pairwise using SDT in this study. Trioza species considered in this study included those from Percy et al. 48 and Khamis et al. 49 .
Tests for Ca. Liberibacter spp.. TaqMan Multiplex Real-Time PCR tests were done on an ABI 7500 Fast Thermocycler (Thermo Fisher Scientific Inc., Waltham, MA) or a SmartCycler II (Cepheid, Sunnyvale, CA) and the DNA extracts were assayed for CLaf, CLam, and CLas as described [50][51][52] . All reactions included a standard set of positive and negative DNA controls, as well as a non-template water control. At a cycle threshold (Ct) of ≤ 37, it was determined that a sample was positive for the presence of a given bacterium.
Ethical standards. This article does not contain any studies with human participants or animals performed by any of the authors. www.nature.com/scientificreports/

Results
Field detection and morphological identification. The altitudes of all locations investigated ranged from 112 to 174 m above sea level (m.a.s.l). The field-collected insects were morphologically identified as D. citri and T. erytreae using previously published features 4,12,18,39,40 , and by comparison with voucher specimens (Fig. 1). Both the TAMUK-CC Entomology Laboratory in Weslaco, Texas, and the University of Environment and Sustainable Development in Somanya, Ghana, now hold voucher specimens of these samples. Four of seven locations had adults D. citri feeding on mature and/or young growing oranges jasmine leaves during the study. We found that D. citri was more established and reasonably widespread in Volta Region than T. erytreae, given the vast dispersion of the positive detection in different locations and the observation of developing nymphs and eggs at three different sites (Table 1).

Molecular detection.
A subset of six adults each of D. citri and T. erytreae were chosen at random to represent the geographical diversity of the sampled insects, and their gene-specific DNA amplicons were found to be of the predicted sizes (Table 2).
There was a total of six sequences for D. citri COI; four using DCITRI COI-L/DCITRI COI-R and two using Te-6U30/Te-720L26. The T. erytreae samples had six sequences. D. citri sequences obtained with primers DCITRI COI-L/DCITRI COI-R showed significant matches (100.0% nt identical; 100% query coverage; E-value 0.0) using BLASTn between these sequences and corresponding gene-specific sequences of D. citri that are deposited in GenBank from different countries. When T. erytreae primers Te-6U30/Te-720L26 were used to amplify the COI gene of D. citri, the results also revealed a significant (99.30-99.58% nt identical; 100% query coverage; E-value 0.0) matches with sequences of D. citri from other countries. Trioza erytreae sequences obtained with primers Te-6U30/Te-720L26 showed a significant (100% nt identical; 100% query coverage; E-value 0.0) matches using BLASTn between these sequences and corresponding gene-specific sequences of T. erytreae that are deposited in GenBank from different countries.

Phylogenetic analysis. Maximum likelihood (ML) phylogenetic analysis of each gene-specific sequence
was performed, and the Tamura 3-parameter model was shown to have the lowest Bayesian Information Criterion (BIC) scores. As expected, it was predicted that the Ghana mtCOI sequences of T. erytreae (OR036870-OR036875) and D. citri (OR036866-OR036869) fall inside the T. erytreae and D. citri clade of the psyllid ML trees (Fig. 2). Our analysis revealed that T. erytreae samples from Ghana clustered with samples from other countries. After further analysis, it was shown that the mtCOI sequences unique to D. citri clustered strongly into the Western clade, which consists of populations from many countries. In addition, the D. citri samples from Ghana may likely represent a separate introduction event into Africa considering how distant they are from the other samples of Benin and Nigeria.

Color-coded pairwise identity. Possible demarcation criteria for the T. erytreae is assigned and compared
to other species of Trioza. Based on the analyzed partial mtCOI sequences, the T. erytreae samples from Ghana  www.nature.com/scientificreports/  www.nature.com/scientificreports/ appear to be distant relatives of the species T. erytreae based on the combination of sequence variation and geographical segregation (Fig. 3). Trioza erytreae collected from Ghana belong to the same demarcation as T. erytreae from other countries when analyzed using the SDT.

Discussion
For the first time, we report the presence of D. citri and T. erytreae in Ghana using both morphological and molecular techniques. However, there have been recent reports of D. citri in Nigeria (West Africa) 10 and other African countries, such as Ethiopia 9 , Tanzania 53 , Kenya 8 and Benin 11 . In response, many citrus-producing areas in eastern and southern African countries have increased the intensity of their pest surveillance and monitoring 10 .
Herein, we initiated this study to ascertain the status of D. citri in Ghana to make early detection (if present) to inform concerted management efforts in Ghana and across sub-Saharan Africa. In addition, preventing an invasion is almost always cheaper than managing an invasive species once it has already entered an area 54 . Moreover, this was done due to the dangers of transporting plants around the world and the fact that citrus has been grown in Ghana for centuries. Diaphorina citri in Ghana was confirmed at elevations of less than 200 m.a.s.l. According to Holford et al. 55 , in Indonesia and Bhutan, high altitudes above 1000 m.a.s.l limit the incidence and occurrence of HLB and D. citri, respectively. The entire country (i.e., Ghana) has an elevation of less than 1000 m.a.s.l. The mean annual temperatures of Ghana range between 24 to 30 °C, though it can be as low as 18 °C in the south and as high as 40 °C in the north 56 . Diaphorina citri prefers warm and dry climates and suitable temperatures for development range between 25 and 28 °C 57 . It can also tolerate temperatures above 40 °C 29 . However, high temperatures may www.nature.com/scientificreports/ decrease the flight capacity of the pest 29 . Average annual rainfall in the north of Ghana is below 1000 mm, whereas it averages approximately 2000 mm in the south 56 . Given the suitable environmental conditions in Ghana for D. citri, it is possible that the pest is widely distributed or can spread to other citrus-growing regions in the country. Trioza erytreae was identified in one location in the Volta Region during the survey. A temperature-based phenology model study on T. erytreae predicted that optimum temperatures for the pest ranged from 20 to 25 °C 58 . The temperature in Ghana suggests that T. erytreae has the potential to spread in the country. However, it will be easier to manage T. erytreae than D. citri in Ghana because of its environmental requirements. In the future, it will be imprudent to ease off in developing management techniques aimed at preventing and managing agricultural pests because of their potential to adapt to the changing climates in many regions 59 .
A recent study, which used a species distribution model of suitability, concluded that most tropical Africa has a suitable climate for spreading D. citri and T. erytreae [5][6][7] . As a result of their research, they created a predictive niche map showing that many West, East, and Central African countries, including Ghana, are at high risk for D. citri establishment. Considering this, the presence of D. citri in Ghana demonstrates the severe threat posed by this invasive species to Ghana and other African countries where the pest is absent. The mtCOI of D. citri has been widely used in genetic variation and population structure studies 60-62 because of its adaptability in diversifying insect populations across different geographical areas.
The presence of D. citri is of particular concern in Ghana, where agriculture is the backbone of the economy. Moreover, D. citri has risen to become a global threat to the viability of citrus businesses wherever the pest and the disease it transmits occur 25 . Agriculture remains a critical tool for sustainable development in many countries across sub-Saharan Africa, providing hundreds of millions of rural poor with new avenues out of poverty through smallholder farming, work in high-value crop production, entrepreneurial endeavors, and employment in rural and non-farming sectors. Despite efforts to ensure sustainable crop production, pests, and diseases persistently pose a threat on the continent. One such pest is T. erytreae, which is limited to Africa, the Middle East, and Europe 5,6 . The presence of D. citri in Ghana, which has a wide distribution in the North, Central, and South America, demonstrates that the combined effects of the HLB vectors could worsen the present losses associated with citrus pests. Trioza erytreae is heat-sensitive and develops best in the cooler highlands, while the Asiatic strain is believed to be more virulent and damaging overall 63 . This, however, suggests that the presence of HLB could threaten the sustainable production of citrus in Ghana due to the suitable climate suitable areas in the country 7 .
Huanglongbing causal agents can be disseminated via grafting and vegetative propagation. However, D. citri is implicated in much of their long-distance and within orchard distribution. Diaphorina citri can be transported over long distances by moving citrus materials like seedlings and alternate host plants. However, the psyllid may also move over long distances, and the prevailing wind direction and intensity can facilitate distance movement [64][65][66] . Diaphorina citri is primarily responsible for the introduction and subsequent spread of the Asian type of HLB in various parts of the world, including Brazil 67 , Texas 68 , China 69 , and California 70 .
In this study, D. citri was obtained from an alternate host plant (M. paniculata). Alternate hosts play a critical role in the dispersal and management of invasive pests 71,72 . Murraya paniculata is grown as an ornamental tree or hedge due to its durability, adaptability to a wide variety of soil, and suitability for larger hedges. In addition, the plant has antimicrobial, antioxidant, red blood cell membrane stabilization, and anti-inflammatory properties and is used to treat many diseases 73 . As a result, it has been used in many parts of the world for symptoms such as nausea, vomiting, constipation, diarrhea, stomach discomfort, headache, fluid retention, and clot formation 16 . In Ghana, the plant is mainly grown for its therapeutic uses, beautification and as a hedge. Implication for management. Although the psyllids were collected from non-citrus host plants in Volta Region, Ghana, nationwide surveys targeting citrus and non-citrus host plants are urgently needed to define the extent of the spread in Ghana. Effective management of psyllids and HLB can only be achieved through a thorough assessment of the distribution of the pest and the disease it transmits. In addition, identifying localities that are free of infestation and where clean nursery programs can be established would offer some level of management against the psyllids. The removal of alternate host plants can be facilitated by a better understanding of areas where these plants are planted, which may also reveal the presence of previously unknown reservoirs of Ca. Liberibacter species. The farming practices in Ghana predominantly revolve around subsistence farming, which contradicts the effective implementation of recommended practices for managing HLB. These practices include establishing clean nurseries, implementing intensive psyllid management across larger contiguous blocks, and more. To effectively manage the psyllids, there is a need for regular inspection of citrus and non-citrus host plants at the Ghanaian ports of entry, and intercepted psyllids should be tested for HLB 5,74 . Given the confirmation of both D. citri and T. erytreae, it is essential to evaluate the existing distribution of D. citri not only in Ghana but also in neighboring regions. Coordinated regional management initiatives should be initiated promptly to eliminate these destructive pests before they become endemic or spread CLas, CLam, and CLaf, which could have severe consequences throughout the region. If found in citrus, efforts will be required to identify optimal crop management, such as crop rotation, intercropping and planting time. Also, developing resistance varieties, such as genetically engineered insecticidal types, could help control the psyllid populations in an environmentally friendly manner because resistance in field populations of D. citri has been reported in citrus groves 75,76 . In this study, it is important to note that D. citri was also sequenced using the T. erytreae-specific primer. This T. erytreae-specific primer used for D. citri sequencing has GenBank accession numbers OR036900 and OR036901. This information is valuable for identifying psyllids at entry points, thereby enhancing detection capabilities. Furthermore, Wenninger et al. 77 reported that D. citri adults can exhibit various colorations, including gray/brown, blue/green, and orange/yellow forms, with noticeable differences in abdominal coloration. To www.nature.com/scientificreports/ facilitate early detection of D. citri, a combination of morphological identification and subsequent molecular studies can be useful in identifying psyllids present on planting materials at entry points.

Data availability
The sequences obtained in this study have been deposited in NCBI GenBank with the following accession numbers: OR036866-OR036875 and OR036900-OR036901. www.nature.com/scientificreports/